Clostridium difficile, an anaerobic organism, is the major causative agent of pseudomembranous colitis (PMC) in humans. PMC is characterized by diarrhea, a severe inflammation of the colonic mucosa, and formation of pseudomembranes that are composed of fibrin, mucus, necrotic epithelial cells, and leukocytes. The pseudomembrane can form a sheath over the entire colonic mucosa. In addition to causing PMC, C. difficile is believed to play a role in other less severe gastrointestinal illnesses; the organism is estimated to cause approximately 25% of reported cases of antibiotic-associated diarrhea. Brettle and Wallace (1984) J. Infect. 8: 123-128; Gilligan et al. (1981) J. Clin. Microbiol. 14: 26-31. Diarrhea affects approximately 25 million persons annually in the U.S. alone, and causes almost 11,000 deaths. Peterson and Kelly (1993) Lab. Diagnosis Infect. Dis. 7: 277-292. C. difficile-caused diseases are not limited to gastrointestinal illnesses, as the organism can cause abscesses, wound infections, osteomyelitis, urogenital tract infections, septicemia, peritonitis, and pleuritis. Lyerly et al. (1988) Clin. Microbiol. Rev. 1: 1-18; Hafiz et al. (1975) Lancet 1: 420-421; Levett (1986) J. Infect. 12: 253-263; Saginur et al. (1983) J. Infect. Dis. 147: 1105. Antibiotics can predispose a host animal to PMC and other C. difficile-related illnesses, as the disturbance of the normal bacterial flora by the antibiotic disrupts the major barrier against colonization by pathogens, rendering the host animal susceptible to colonization by pathogens such as C. difficile. Hospitals and chronic care facilities are significant sources of C. difficile infection, with one study finding that 21% of patients acquired C. difficile infection during hospitalization. McFarland et al. (1989) N. Engl. J. Med. 320: 204.
The high frequency of C. difficile infection, coupled with the likelihood of a poor clinical outcome for cases that are not treated promptly, makes clear the need for rapid and accurate tests to detect C. difficile infection, determine whether any C. difficile present is toxigenic, and evaluate the effectiveness of treatment. Previously available methods for detecting C. difficile are much less than optimal for effective diagnosis and treatment of infection. One previously known method for detecting C. difficile infection is culture on agar media. The efficacy of this method is hampered by the significant variation in results that are obtained using different media, and the high rate of false positives (10-29%). Peterson and Kelly, supra. An additional disadvantage of this method is the lengthy culture period required before visible C. difficile colonies are discernable. A commercially available assay for C. difficile involves latex agglutination of an antigen that was eventually identified as C. difficile glutamate dehydrogenase. Lyerly et al. (1991) J. Clin. Microbiol. 29: 2639; Lyerly et al. (1986) J. Clin. Microbiol. 23: 622. However, this assay suffers from widely varying and insufficient sensitivity and specificity, with sensitivity ranging from 68% to 93% and specificity between 80% and 95%. Peterson and Kelly, supra. Moreover, previously available glutamate dehydrogenase assays were not thought to be useful for detection of toxigenic strains of C. difficile because glutamate dehydrogenase is produced by non-toxigenic strains of C. difficile, as well as toxigenic strains.
Non-toxigenic strains of C. difficile are generally considered clinically insignificant, while toxigenic strains can be lethal. Although distinguishing between toxigenic and non-toxigenic C. difficile strains is thus of great importance, previously known assays that were used in attempts to accomplish this goal were ineffective. One commonly used diagnostic method for detecting the presence of toxigenic C. difficile involves determining whether C. difficile is cytotoxic to susceptible cell lines. This cytotoxicity is the result of either or both of the two toxins produced by C. difficile, an enterotoxin designated toxin A and a cytotoxin designated toxin B, both of which are believed to be involved in the pathogenesis of PMC. The cytotoxicity assay has significant drawbacks for clinical use however, including the need to maintain tissue culture lines and the relatively low sensitivity of the assay. For example, Peterson and Kelly, supra., found that the sensitivity of cytotoxin detection alone ranged from 67% to 100%, and other researchers found sensitivity to be as low as 71%. Demlee et al. (1985) J. Clin. Microbiol. 21: 323. Immunoassays for toxins A and/or B have also been used to detect C. difficile in samples, but these methods suffer from low sensitivity (63% to 88%). Peterson and Kelly, supra.
Thus, a need exists for assays to detect the presence of C. difficile in a sample that are rapid, sensitive, specific, and cost-effective. Assays to determine whether an infecting C. difficile strain is toxigenic are also needed. The present invention fulfills these and other needs.